Method of treating patients with diabetes

ABSTRACT

The present invention is directed to a method of treating a patient with diabetes by implanting an improved macrocapsule containing living islet cells in the patient. The improved macrocapsules encapsulate microcapsules containing islet cells, to make the system more biocompatible by decreasing the surface area and surface roughness of microencapsulated biological materials; increasing stability of microencapsulated islet cells; enhancing cytoprotectivity by increasing diffusion distance of encapsulated islet cells from cytotoxins secreted in vivo; providing retrievability of microencapsulated material; and providing a system of sustained release of the cellular products.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.08/473,018, filed Jun. 6, 1995, now U.S. Pat. No. 5,788,988, which is inturn a divisional of U.S. application Ser. No. 08/400,442, filed Mar. 6,1995, now U.S. Pat. No. 5,545,423, which is in turn a continuation ofU.S. application Ser. No. 07/981,852, filed Nov. 24, 1992, nowabandoned, which is in turn a continuation-in-part of U.S. applicationSer. No. 07/797,704, filed Nov. 25, 1991, now abandoned.

FIELD OF THE INVENTION

The present invention relates to a new form of biocompatible materialswhich envelop encapsulated or free cells to provide an immune barrier.The resulting encapsulated material is generally small, but macroscopic,so that it is retrievable in situ. More specifically the presentinvention relates to a composition and system for treatment of diabetes.

BACKGROUND OF THE INVENTION

Diabetes Mellitus is a serious disease afflicting over 100 millionpeople worldwide. In the United States, there are more than 12 milliondiabetics, with 600,000 new cases diagnosed each year.Insulin-dependent, or Type I diabetics, require daily injections ofinsulin to prevent them from lapsing into coma.

With the discovery of insulin in 1928, it was thought that diabetes hadbeen cured. Unfortunately, despite insulin therapy, the majorcomplications of the disease caused by high blood sugar levels persist.Each year, diabetes accounts for 40,000 limb amputations and 5,000 newcases of blindness in the United States. Among teenagers, diabetes isthe leading cause of kidney failure. Data from the National Institutesof Health show that the rate of heart disease and stroke is twice asgreat in diabetics than in the general population.

The diabetic patient faces a 30% reduced lifespan. Multiple insulininjections given periodically throughout the day cannot duplicate theprecise feedback of insulin secretion from the pancreas. The onlycurrent method of achieving minute-to-minute glucose control is bypancreas transplantation.

While whole organ pancreas transplantation represents a significantadvance in diabetes therapy, the operation is technically difficult, oflimited success and use because of the problems of rejection, and itstill presents a significant risk to the patient. An attractivealternative would be to extract the insulin-producing cells (islets)from a donor pancreas and to inject these cells into the diabeticpatient, thus effecting a cure. The use of such cells, however, wouldstill run the risk of rejection by the host.

Microencapsulation of islets by an alginate-PLL-alginate membrane (i.e.,an alginate-poly-L-lysine-alginate membrane) is a potential method forprevention of rejection by the host's immune system. By this technique,researchers are able to encapsulate living islets in a protectivemembrane that allows insulin to be secreted, yet prevents antibodiesfrom reaching the islets, causing rejection of the cells. This membrane(or microcapsule) protects the islet from rejection and allows insulinto be secreted through its "pores" to maintain the diabetic in normalglucose control

Successful transplants of microencapsulated islets have not beenclinically feasible to date due to fundamental problems of transplantrejection and/or a fibrotic reaction to the microcapsule membrane. Limand Sun, 1980, reported the first successful implantation ofmicroencapsulated islets and described normalization of blood sugar indiabetic rats. However, for microencapsulated islets to be clinicallyuseful and applicable in humans, it is important that theimmunoprotective membrane be biocompatible, allow adequate diffusion forthe encapsulated cells to respond appropriately to a stimulatory signal,provide the encapsulated cells with necessary nutrients, and beretrievable. Retrievability is desirable for a variety of reasons, e.g.,so that accumulation of the implanted materials can be avoided, so thatencapsulated cells can be removed from the recipient when no longerneeded or desired (e.g., when the product(s) of the encapsulated cellsare no longer needed, if the encapsulated cells fail to perform asdesired, etc.), so that encapsulated cells can be removed if/when theybecome non-viable, and the like. Currently there are no reports ofsuccessful reversal of diabetes in humans by transplantation ofencapsulated islets.

Biocompatibility of encapsulated islets remains a fundamental problem.The term "biocompatible" is used herein in its broad sense, and relatesto the ability of the material to result in long-term in vivo functionof transplanted biological material, as well as its ability to avoid aforeign body, fibrotic response. A major problem with microencapsulationtechnology has been the occurrence of fibrous overgrowth of theepicapsular surface, resulting in cell death and early graft failure.Despite extensive studies, the pathological basis of this phenomenon inalginate based capsules remains poorly understood. However, severalfactors have recently been identified as being involved in graftfailure, e.g., the guluronic acid/mannuronic acid content of thealginate employed, imperfections in the microcapsule membrane (allowingexposure of poly-L-lysine to the in vivo environment), failure of themicrocapsule membrane to completely cover the cells being encapsulated(thereby allowing exposure of the cells to the in vivo environment), andthe like.

Alginate is a polysaccharide isolated from marine brown algae includingLaminaria hyperborea, Laminaria digitata, Ascophyllum nodosum andMacrocystis pyrifera. Alginate forms ionically crosslinked gels withmost di- and multivalent cations. Calcium cations are most widely used,and give rise to a three-dimensional network in the form of an ionicallycrosslinked gel by inter-chain binding between G-blocks. (Skjåk-Braek,1988).

It has recently been demonstrated that the mannuronic acid residues arethe active cytokine inducers in alginate, and since these cytokines(IL-1 and TNF) are known to be potent stimulators of fibroblastproliferation (Otterlei et al., 1991), it was deduced that alginatecapsules high in mannuronic acid content (M-content) were responsible inpart for the fibrotic response reported in the past (Soon-Shiong et al.1991). More significantly, it has been found that this reaction could beameliorated by increasing the guluronic acid content (G-content) of thealginate capsule since guluronic acid appears not to beimmunostimulating. Furthermore, it has been demonstrated thatcyclosporin A resulted in a dose dependent inhibition of mannuronicacid-induced TNF and IL-1 stimulation of human monocytes in vitro. Basedon this information, it has been hypothesized that the fibrotic reactionof the microcapsule could be ameliorated in part by an alginateformulation high in guluronic acid content, as well as a subtherapeuticcourse of cyclosporin A to inhibit cytokine stimulation. By this method,diabetes in the spontaneous diabetic dog model has been successfullyreversed by transplantation of donor islets encapsulated in highG-content alginate (Soon-Shiong et al. 1991).

Polyethylene glycols (PEG; also referred to as polyethylene oxide, PEO)have been investigated extensively in recent years for use asbiocompatible, protein repulsive, noninflammatory, and nonimmunogenicmodifiers for drugs, proteins, enzymes, and surfaces of implantedmaterials. The basis for these extraordinary characteristics has beenattributed to the flexibility of the polymer backbone, and the volumeexclusion effect of this polymer in solution or when immobilized at asurface. The solubility of PEGs in water, as well as a number of commonorganic solvents, facilitates modification by a variety of chemicalreactions. A recent review (Harris, 1985) describes the synthesis ofnumerous derivatives of PEG and the immobilization thereof to surfaces,proteins, and drugs.

PEG bound to bovine serum albumin has shown reduced immunogenicity andincreased circulation times in a rabbit (Abuchowski et al., 1977). Drugssuch as penicillin, aspirin, amphetamine, quinidine, procaine, andatropine have been attached to PEG in order to increase their durationof activity as a result of slow release (Weiner et al., 1974; Zalipskyet al., 1983). PEG covalently bound to poly-L-lysine (PLL) has been usedto enhance the biocompatibility of alginate-PLL microcapsules used forthe encapsulation of cells. PEG has been covalently bound topolysaccharides such as dextran (Pitha et al., 1979, Duval et al.,1991), chitosan (Harris et al., 1984) and alginates (Desai et al.,1991). These modifications confer organic solubility to thepolysaccharides.

Surfaces modified with PEG were found to be extremely nonthrombogenic(Desai and Hubbell, 1991a; Nagoaka and Nakao, 1990); resistant tofibrous overgrowth in vivo (Desal and Hubbell, 1992a) and resistant tobacterial adhesion (Desai and Hubbell, 1992b). Solutions containing PEGhave also been found to enhance the preservation of organs fortransplantation (Collins et al.; Zheng et al., 1991). The basis of thepreservation activity is not clearly understood but has been attributedto adhesion of PEG to cell surface molecules with a resultant change inthe presentation of antigen so as to alter the nature of the immuneresponse.

Crosslinked PEG gels have been prepared and utilized for immobilizationof enzymes and microbial cells. Fukui and Tanaka (1976) and Fukui etal., (1987) have prepared polymerizable derivatives of PEG (such as thedimethacrylate) and photocrosslinked them with UV light in the presenceof a suitable initiator to form a covalently crosslinked gel. Kumakuraand Kaetsu (1983) have reported the polymerization and crosslinking ofdiacrylate derivatives of PEG by gamma radiation for the purpose ofimmobilizing microbial cells. Due to the mild nature of thephotopolymerization, i.e., absence of heating, without shifting pH toextreme values, and without the use of toxic chemicals, the Fukui andTanaka (1976) publication suggests that this technique is desirable forthe entrapment not only of enzymes, but also for cells and organelles.

Dupuy et al. (1988) have recently described a photopolymerizationprocess for the entrapment of agarose embedded pancreatic islets inmicrospheres of crosslinked acrylamide. However, this referencedescribes entrapment of individual microspheres, but does not describefurther entrapment of these already entrapped cells. Visible light wasused as the initiating radiation in the presence of a photochemicalsensitizer (vitamin B2, i.e., riboflavin), and a cocatalyst(N-N-N'-N'-tetramethylethylene diamine). A high pressure mercury lampwas used as the source of visible radiation and the islets weredemonstrated to maintain a good viability in vitro following thepolymerization step.

Visible radiation between wavelengths of 400-700 nm have been determinedto be nontoxic to living cells (Karu, 1990; Dupuy et al., 1988). Arecent review (Eaton, 1986) describes a variety of dyes and cocatalyststhat may be used as polymerization initiators in the presence ofappropriate visible radiation.

In recent years considerable interest has been expressed in the use oflasers for polymerization processes (Wu, 1990). These polymerizationsare extremely fast and may be completed in milliseconds (Decker andMoussa, 1989; Hoyle et al., 1989; Eaton, 1986). The use of coherentradiation often results in the polymerization being innocuous to livingcells. This arises from the use of wavelength specific chromophores asthe polymerization initiators, and these chromophores are typically theonly species in the polymer/cell suspension that absorb the incidentradiation.

SUMMARY OF THE INVENTION

It has previously been demonstrated that Type I insulin-dependentdiabetes can be reversed in rats and dogs by implantation ofmicro-encapsulated pancreatic islets in the peritoneal cavity of theseanimals using both allograft (dog islets to dog recipients) andxenograft (dog islets to rat recipients) models (Soon-Shiong et al.,1991). The alginate microcapsules employed in these studies provideimmunoprotectivity, and result in graft survival of allografts forseveral months in the absence of immunosuppression. The alginate gels,however, are ionically crosslinked and, therefore, are subject tobreakage and resorption as a result of ionic equilibration in vivo. Inaddition, free microcapsules are difficult to retrieve due to theirsmall size (200-600 μm) and nonlocalization within the peritonealcavity.

While it is known that the mannuronic acid--guluronic acid (M-G) ratioof the alginate employed plays an important role in preventing fibrosis,in accordance with the present invention, other factors involvingvarious defects in prior art microcapsules have been identified whichplay an important role in preventing long term viability of thetransplanted graft. Photo-micrographs demonstrate these heretoforeunrecognized defects in prior art microcapsules.

In accordance with the present invention, a number of factors relatingto capsule failure, i.e., likelihood of inducing an immune response,likelihood of loss of function due to death of the encapsulated cells,the degree of protection afforded encapsulated cells, and the like, havebeen identified, i.e., that:

(i) Both mechanical and chemical stability of the microcapsule play acritical role in fibrous overgrowth. It has been discovered that thewater-soluble membrane dissolves over a prolonged period of in vivoexposure, eventually exposing the encapsulated material to the host'simmune system, initiating a rejection response;

(ii) The "roughness" of the capsule membrane, as well as defectivemicrocapsules, result in macrophage activation, cell adherence, cellularovergrowth and eventual fibrosis;

(iii) Disruption or breakage of microcapsules, with cracks in thecapsule membrane, results in fibrosis;

(iv) While alginate, specifically high G-content alginate, is relativelybiocompatible, it has been discovered that poly-L-lysine (PLL) is apotent stimulator for fibrous overgrowth in vivo. Indeed, the prior artdescribes attempts to cover the outer coat of PLL with an outer layer ofalginate to prevent this overgrowth problem. However, according to thepresent invention, it has been discovered that any disruption of themembrane, or any imperfection of coating of the membrane results infibrous overgrowth when polymer materials such as PLL are exposed.Furthermore, current methods of covering PLL with an outer layer ofalginate are ineffective to fully prevent some exposure of PLL overtime, in vivo; and

(v) Exposure of biologically active material on the surface of themicrocapsule (because such material is not adequately entrapped in thegel forming the microcapsule) exposes such material to the host's immunesystem, initiating an immune response.

In accordance with the present invention, it has been further discoveredthat individual spherical microcapsules provide a large exposed surfacearea, facilitating transport of nutrients through the membrane, while onthe other hand, increasing the probability of cell adherence and fibrousovergrowth. It has been found that by reducing this exposed microcapsulesurface area and/or by reducing exposure of any unbound, positivelycharged polylysine, while maintaining the critical diffusion capacity ofthe immunoprotective membrane, increased biocompatibility will ensue. Byreduction of exposed microcapsule surface area, the percentage of"roughness" associated with each individual microcapsule would bereduced, resulting in improved biocompatibility. In addition, inaccordance with the present invention, it has been recognized thatimproving mechanical integrity of the capsule is an important step inachieving long term graft function.

In accordance with the present invention, it has been found that longterm graft function can be achieved by entrapping or encasingbiologically active material, optionally contained within a microcapsule(e.g., individual microencapsulated cells) in a macrocapsule which isbiocompatible, thereby (i) increasing the cytoprotectivity of theentrapped individually encapsulated cells, (ii) reducing exposure ofunbound positively charged polylysine to the host in vivo environment,(iii) enhancing the mechanical stability of the capsular membrane, (iv)reducing the exposed microcapsule surface area roughness; and (v)reducing the exposure of cells adhering to the surface of themicrocapsule to the host in vivo environment; all of the aboveadvantages are obtained while (vi) maintaining the diffusion capacity ofthe polymeric material used for encapsulation, thereby allowing theentrapped encapsulated cells to be nourished and respond to astimulatory signal.

Additionally, macrocapsules of the present invention provide a system ofrapid but sustained release of the material made by and secreted by theencapsulated cell(s), which in turn provides for more regulated controlof physiological processes (e.g., blood glucose levels in the case ofencapsulated islets). Specifically, in response to an intravenousglucose stimulus, insulin release occurs more rapidly from encapsulatedislets entrapped within a macrocapsule than from free floatingmicroencapsulated islets, as demonstrated by the in vivo intravenousglucose stimulation studies described in Example 9, below. In addition,insulin release from these gel entrapped microcapsules is sustained overa longer period of time, as demonstrated by the in vitro glucosestimulation studies described in Example 8, below.

The present invention also permits retrievability of the implant becauseof its macroscopic size and its grouping of the variousmicroencapsulated islets into a single package or a plurality ofmacroscopic packages. Retrievability is desirable for a variety ofreasons, e.g., so that accumulation of the implanted materials can beavoided, so that encapsulated cells can be removed from the recipientwhen no longer needed or desired (e.g., when the product(s) of theencapsulated cells are no longer needed, if the encapsulated cells failto perform as desired, etc.), so that encapsulated cells can be removedif/when they become non-viable, and the like.

The present invention overcomes the problems of the prior art byimproving the cytoprotectivity and biocompatibility of implantedbiological systems. The present invention also provides a gel entrapmentsystem which provides a rapid response of entrapped cells to astimulatory signal. The present invention further provides a sustainedrelease system made from microencapsulated cells which are furtherpackaged in a macrocapsule. The present invention also provides a systemthat localizes microcapsules in a particular region (e.g., a region ofhigh vascularization such as the omentum) as well as a system whichminimizes the breakage of microcapsules, and facilitates their readyretrieval.

As will be readily apparent to a person of skill in the art, themacrocapsules of the present invention may be used to entrap not onlyencapsulated or unencapsulated living cells, but also any chemicalreagents which may have a pharmacological or physiological effect uponsustained release by the system disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing of one embodiment of a macrocapsule of the presentinvention containing encapsulated biological material.

FIG. 2 is a graph of the blood glucose of rats implanted with entrappedencapsulated islets according to the present invention (filled circle), free-floating encapsulated islets (filled triangle ▴) andunencapsulated islets (open circle ∘) over time, when the islets arexenografts (canine islets).

FIG. 3 is a graph of the serum glucose response to an intravenousglucose challenge (IVGTT) in rats treated with macroencapsulated canineislets according to the present invention, compared to the response inrats transplanted with free floating microencapsulated canine islets 7days after implantation of the microcapsules and macrocapsules.

FIG. 4 is a graph of the serum glucose response to an intravenousglucose challenge (IVGTT) in rats treated with macroencapsulated canineislets according to the present invention, compared to the response inrats transplanted with free floating microencapsulated canine islets.

FIG. 5 is a graph of blood glucose levels of rats implanted withentrapped encapsulated islets according to the present invention (filledcircle ) over time, when the islets are xenografts (canine islets). Onday 14, the entrapped encapsulated islets were retrieved. The diabeticstate recurred within 24 hours following retrieval of the entrappedencapsulated islets, proving the viability of the islets entrappedwithin the macrocapsule of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The following specification describes materials and methods ofmacroencapsulating biologically active materials, such as living cells,to provide an enhanced biocompatible, retrievable system which isuseful, for example, for cell containment and transplantation. In thefollowing description, numerous details such as specific materials andmethods are set forth in order to provide a more complete understandingof the present invention. It is understood by those skilled in the artthat the present invention can be practiced without these specificdetails. In other instances, well known materials and methods are notdescribed in detail so as not to obscure the present invention. Inaddition, the following description is given with particular referencein many instances to islets and diabetes treatment. This description isgiven as a presently preferred application of the present invention.Nonetheless, it is apparent that the present invention is not limited tothe treatment of a single disease state. The present invention can beused equally as well for other cell types and treatment of otherdisorders or for other physiological purposes and could be used withmicroencapsulated cells or unencapsulated cells of any type.

As part of the work carried out pursuant to the present invention,photomicrographs were taken of prior art alginate-PLL-alginatemicrocapsules in efforts to identify the faults and defects therein(see, for example, Clayton, in J. Microencapsulation 8:221-233 (1991)and Wijsman et al., in J. Transplantation 54:588-592 (1992) for adescription of prior art microcapsules). The resulting micrographsreadily reveal the faults and defects in prior art microcapsules. Forexample, a photomicrograph (40×) of an empty alginate-PLL-alginatemicrocapsule (retrieved from the peritoneal cavity of a rat)demonstrates that cellular overgrowth (appearing as a darkened area)occurs in areas of the exposed microcapsule gel or in areas of exposedpolylysine when capsular membrane integrity is lost. A photomicrograph(100×) of an alginate-PLL-alginate encapsulated islet shows areas of"roughness" or "stress marks" associated with individual microcapsules.The surface area of such individual microcapsules is large and thus inturn, the exposure of "rough" surfaces is huge, increasing the risk ofcellular overgrowth. In addition, imperfect covering of polylysine by anouter coat of alginate results in potential overgrowth.

A photomicrograph (40×) of a defective empty microcapsule, amongstintact empty alginate microcapsules, all retrieved at the same time fromthe peritoneal cavity of a rat, demonstrates cellular overgrowth (whichappears as darkened areas). Vigorous overgrowth is observed to beassociated only with the defective capsule. This provides furtherevidence that minor imperfections invoke a foreign body response todefective capsules and to exposed capsular material, especially PLL.

A photomicrograph (40×) of disrupted alginate microcapsules containingcanine islets (transplanted free floating into the peritoneal cavity),retrieved from the peritoneal cavity of a diabetic dog when reversal ofdiabetes failed, shows the presence of disrupted capsules, providingevidence that loss of mechanical stability plays an important role ingraft failure. These micro-encapsulated canine islets successfullyreversed diabetes, but only for a short period.

As used herein, the term "macrocapsule" means a capsule of gel materialsurrounding biologically active material, optionally contained within amicrocapsule (e.g., one or more microcapsules, where each microcapsulecontains at least one living cell, such as islets or otherpharmacological agent producing cells, or certain drugs orphysiologically active agents). The term "macrocapsule" may include"macro-membranes," "macrogels," "gel entrapped microcapsules," "lace,""noodles," "teabags," "threads," "worms," and the like, which a personof skill in the art would understand refers to the general class ofcompositions described herein. While the actual dimensions of thevarious components of the invention particles are not critical, the term"microcapsules" is generally used to refer to particles wherein thelargest dimensions thereof fall in the range of about 5 up to 4000microns, while the term "macrocapsules" is generally used to refer toparticles wherein the largest dimensions thereof fall in the range ofabout 500 microns up to about 50 cm. What is important according to thepresent invention is that the contents of a microcapsule be furtherencapsulated into a macrocapsule, thereby affording the added benefitsof the macroencapsulating polymer, as described herein.

In accordance with the present invention, biologically active material,e.g., a plurality of cell-containing microcapsules, are covered with athick layer of gelled material, forming a microcapsule-containingmacrocapsule. A schematic showing a cross-section of such a macrocapsuleis provided in FIG. 1. Inspection of the Figure reveals that themacrocapsule itself contains no polycation or other fibrogenic surface;instead, the immunoprotective membrane (i.e., polycation such as PLL) islocalized at the surface of the microcapsules, which are buried deepwithin the macrocapsule (and protected from exposure to the in vivoenvironment by thousands of molecular layers). The core of themicrocapsules (as well as the macrocapsule itself) are maintained in thegelled state (i.e., crosslinked or insoluble form), in contrast to priorart encapsulation systems, wherein the core material is liquefied.

Generally the layer of gelled material in invention macrocapsules has athickness of at least about 1 micron, with a thickness of at least about20-40 microns being preferred, and a thickness of at least about 50microns or greater being especially preferred. This provides moreeffective masking of the polycation layer on the microcapsule than thefew molecule thickness (of alginate outer layer) achieved in thepreparation of prior art microcapsules. The layer of gelled material ininvention macrocapsules prevents direct exposure of any immunogenicagents at the microcapsule surface (e.g., polycations, unencapsulatedcells, and the like) to the in vivo environment, thereby preventingimmune response triggered by prior art microcapsules. In addition, thelayer of gelled material is exceptionally stable to long-term exposureto physiological conditions since the gelled material is ionicallyand/or covalently crosslinked to itself, and does not depend on aninteraction with the incorporated material for strength and/orstability. This is in direct contrast with alginate outer layersemployed in prior art microcapsules, wherein the sole means of anchorageof the alginate outer layer to the capsule is the formation of a chargecomplex (i.e., ionic interaction) between the alginate and thepolycation immunoprotective layer.

In a preferred embodiment of the present invention, when microcapsulesare incorporated into macrocapsules, the microcapsules (and themacrocapsules themselves) are maintained in the gelled state (i.e.,ionically and/or covalently crosslinked). This is contrary to theteachings of the prior art, wherein the encapsulating matrix of themicrocapsule is liquefied prior to implantation. In accordance with thepresent invention, it has surprisingly been found that the diffusion ofnutrients in, and products of the encapsulated cells out of inventionmacrocapsules is highly efficient.

Macrocapsules of the invention can be produced in a variety of shapes,i.e., in the shape of a cylinder (i.e., a geometrical solid generated bythe revolution of a rectangle about one of its sides), a sphere (i.e., asolid geometrical figure generated by the revolution of a semicirclearound its diameter), a disc (i.e., a generally flat, circular form), aflat sheet (i.e., a generally flat polygonal form, preferably square orrectangular), a wafer (i.e., an irregular flat sheet), a dog-bone (i.e.,a shape that has a central stem and two ends which are larger indiameter than the central stem, such as a dumbbell), or the like.

The materials used to provide such entrapment could be alginate(preferably high G-content alginate), or a modification of such alginateto improve its biocompatibility and stability, e.g., a polymerizablealginate allowing covalent crosslinkage, or crosslinkable orpolymerizable, water soluble polyalkylene glycol, or combinations ofthese materials. The process to cause gel entrapment of such materialscan be accomplished either by ionic or covalent crosslinkage.

Macrocapsules contemplated for use in the practice of the presentinvention contain therein biologically active material, wherein saidbiologically active material is optionally contained withinmicrocapsules (i.e., a plurality of microcapsules within saidmacrocapsule). The invention macrocapsule can be prepared from a varietyof polymeric materials, such as, for example, covalently crosslinkableor polymerizable linear or branched chain PEG, mixtures of differentmolecular weight covalently crosslinkable or polymerizable linear orbranched chain PEGs, ionically crosslinkable alginate, combinations ofalginate and covalently crosslinkable or polymerizable PEG and modifiedalginate that is capable of being covalently and ionically crosslinked,and the like.

Macrocapsules prepared in accordance with the present invention comprisebiologically active material encapsulated in the above-describedbiocompatible crosslinkable material, wherein the macrocapsule has avolume in which the largest physical dimension is greater than 1 mm.Macrocapsules can contain "free" (i.e., unmodified by any coating) cellsor groups of cells therein. Alternatively, macrocapsules may containcells or groups of cells which are themselves encapsulated withinmicrocapsules.

Biologically active materials contemplated for encapsulation (to producemicrocapsules and/or macrocapsules) according to the present inventioninclude individual living cells or groups of living cells, biologicalmaterials (for diagnostic purposes, e.g., for in vivo evaluation of theeffects of such biological materials on an organism, and conversely, theeffects of the organism on the materials), tumor cells (for evaluationof chemotherapeutic agents), human T-lymphoblastoid cells sensitive tothe cytopathic effects of HIV; pharmacologically active drugs;diagnostic agents, and the like. As employed herein, the term "livingcells" refers to any viable cellular material, regardless of the sourcethereof. Thus, virus cells, prokaryotic cells, and eukaryotic cells arecontemplated. Specifically contemplated cells include islets ofLangerhans (for the treatment of diabetes), dopamine secreting cells(for the treatment of Parkinson's disease), nerve growth factorsecreting cells (for the treatment of Alzheimer's disease), hepatocytes(for treatment of liver dysfunction), adrenaline/angiotensin secretingcells (for regulation of hypo/hypertension), parathyroid cells (forreplacing thyroid function), norepinephrine/metencephalin secretingcells (for the control of pain), hemoglobin (to create artificialblood), and the like.

Covalently crosslinkable and/or polymerizable polyethylene glycols(PEGs) contemplated for use in the practice of the present inventioninclude linear or branched chain PEGs (including STAR PEGs) modifiedwith a substituent X which is capable of undergoing free radicalpolymerization (X is a moiety containing a carbon-carbon double bond ortriple bond capable of free radical polymerization; and X is linkedcovalently to said PEG through linkages selected from ester, ether,thioether, disulfide, amide, imide, secondary amines, tertiary amines,direct carbon-carbon (C--C) linkages, sulfate esters, sulfonate esters,phosphate esters, urethanes, carbonates, and the like). Examples of suchcovalently crosslinkable polyethylene glycols include vinyl and allylethers of polyethylene glycol, acrylate and methacrylate esters ofpolyethylene glycol, and the like.

PEGs having a wide range of molecular weights can be employed in thepractice of the present invention, thus mixtures of different molecularweights of covalently crosslinkable PEGs contemplated for use in thepractice of the present invention include PEGs having a MW in the rangeof about 200 up to 1,000,000 (with molecular weights in the range ofabout 500 up to 100,000 preferred, and PEGs having molecular weights inthe range of about 1000 to 50,000 being the presently most preferred).Such PEGs can be linear or branched chain (including STAR PEGs). STARPEGs are molecules having a central core (such as divinyl benzene) whichis anionically polymerizable under controlled conditions to form livingnuclei having a predetermined number of active sites. Ethylene oxide isadded to the living nuclei and polymerized to produce a known number ofPEG "arms", which are quenched with water when the desired molecularweight is achieved. Alternatively, the central core can be anethoxylated oligomeric glycerol that is used to initiate polymerizationof ethylene oxide to produce a STAR PEG of desired molecular weight.

Alginates contemplated for use in the practice of the present inventioninclude high G-content alginate, high M-content alginate, sodiumalginate, and the like; covalently crosslinkable alginates contemplatedfor use in the practice of the present invention include alginatesmodified with a substituent X which is capable of undergoing freeradical polymerization (X is a moiety containing a carbon-carbon doublebond or triple bond capable of free radical polymerization; and X islinked covalently to said alginate through linkages selected from ester,ether, thioether, disulfide, amide, imide, secondary amines, tertiaryamines, direct carbon-carbon (C--C) linkages, sulfate esters, sulfonateesters, phosphate esters, urethanes, carbonates, and the like). Examplesof covalently crosslinkable alginates include allyl and vinyl ethers ofalginate, acrylate and methacrylate esters of alginate, and the like.

Combinations of alginate (ionically and/or covalently crosslinkable) andcovalently crosslinkable PEG contemplated for use in the practice of thepresent invention include combinations of any two or more of theabove-described alginates and PEGs.

A small amount of a comonomer can optionally be added to thecrosslinking reaction to increase the polymerization rates. Examples ofsuitable comonomers include vinyl pyrrolidinone, acrylamide,methacrylamide, acrylic acid, methacrylic acid, sodium acrylate, sodiummethacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate (HEMA),ethylene glycol diacrylate, ethylene glycol dimethacrylate,pentaerythritol triacrylate, pentaerythritol trimethacrylate,trimethylol propane triacrylate, trimethylol propane trimethacrylate,tripropylene glycol diacrylate, tripropylene glycol dimethacrylate,glyceryl acrylate, glyceryl methacrylate, and the like.

Free radical polymerization of the above-described modified materialscan be carried out in a variety of ways, for example, initiated byirradiation with suitable wavelength electromagnetic radiation (e.g.,visible or ultraviolet radiation) in the presence of a suitablephotoinitiator, and optionally, cocatalyst and/or comonomer.Alternatively, free radical polymerization can be initiated by thermalinitiation by a suitable free radical catalyst.

A variety of free radical initiators, as can readily be identified bythose of skill in the art, can be employed in the practice of thepresent invention. Thus, photoinitiators, thermal initiators, and thelike, can be employed. For example, suitable UV initiators include2,2-dimethoxy-2-phenyl acetophenone and its water soluble derivatives,benzophenone and its water soluble derivatives, benzil and its watersoluble derivatives, thioxanthone and its water soluble derivatives, andthe like. For visible light polymerization, a system of dye (also knownas initiator or photosensitizer) and cocatalyst (also known ascosynergist, activator, initiating intermediate, quenching partner, orfree radical generator) are used. Examples of suitable dyes are ethyleosin, eosin, erythrosin, riboflavin, fluorscein, rose bengal, methyleneblue, thionine, and the like; examples of suitable cocatalysts aretriethanolamine, arginine, methyldiethanol amine, triethylamine, and thelike.

Microcapsules contemplated for use in the practice of the presentinvention can be formed of a variety of biocompatible gel materials,such as, for example, alginate, covalently crosslinkable alginate (i.e.,modified alginate that is covalently and ionically crosslinkable),covalently crosslinkable PEG, combinations of any of the above-describedalginates and any of the above-described PEGs, as well as the optionalpresence of one or more comonomers, as described above.

Microcapsules employed in the practice of the present invention areoptionally treated with an immunoprotective coating, as described by Limin U.S. Pat. No. 4,352,883, incorporated by reference herein (Lim refersto this immunoprotective coating as a "permanent semi-permeablemembrane"). Immunoprotective materials contemplated for use in thepractice of the present invention include polycations (such as polyaminoacids (e.g., polyhistidine, polylysine, polyornithine, and the like);polymers containing primary amine groups, secondary amine groups,tertiary amine groups or pyridinyl nitrogen(s), such aspolyethyleneimine, polyallylamine, polyetheramine, polyvinylpyridine,and the like. Treatment with such immunoprotecting materials can becarried out in a variety of ways, e.g., by crosslinking surface layersof an alginate gelled core containing encapsulated cells with polymershaving acid-reactive groups such as amine or imine groups. This istypically done in a dilute solution of the selected immunoprotectingpolymer. Within limits, semipermeability of the immunoprotecting coatingcan be controlled by proper selection of the molecular weight of theimmunoprotecting polymer, its concentration, and the degree ofcrosslinking with the underlying gel. Molecular weight of theimmunoprotecting polymer can also vary, depending on the degree ofpermeability desired. Typically molecular weights will fall betweenabout 1,000 and 100,000 or higher. Presently preferred immunoprotectingpolymers employed in the practice of the present invention fall in therange of about 10,000 up to 50,000. Optional treatment withimmunoprotective coating can be avoided especially when themacrocapsular or microcapsular material is covalently crosslinkable, sothat the desired porosity (or immunoprotectivity) may be achievedthrough judicious selection of the polymerizable macromonomers employed(i.e., polymerizable alginate, polymerizable PEG, and the like), as wellas mixtures of such monomers.

In a presently preferred embodiment of the present invention, multiplealginate-PLL-alginate microcapsules are entrapped in an alginatemacrocapsule. The microcapsules employed preferably have solid gelcores. The alginate employed can be ionically and/or covalentlycrosslinkable. With modified, covalently crosslinkable alginate, theintroduction of polycations to control microcapsule porosity and/or toprovide an immunoprotecting barrier is optional because the desiredporosity (or immunoprotectivity) may be achieved by using suitableconcentrations of modified alginate or by using modified alginate havingdifferent degrees of substitution with the moiety X, or combinations ofsuch modified alginate.

Another presently preferred embodiment of the invention involves thephotopolymerization of an aqueous physiological covalently crosslinkablelinear or branched chain PEG (e.g., a PEG-diacrylate solution, orpolymerizable equivalent) containing suspended alginate microcapsules(e.g., alginate-PLL-alginate). The microcapsules employed preferablyhave solid gel cores. The appropriate free radical initiators andcocatalysts are used with visible light sources such as a laser or amercury lamp. A solid (i.e., non-liquefied) gel of PEG is formed aroundthe alginate microcapsules which in addition to providing mechanicalsupport and retrievability, forms a nonfibrosing, cell nonadherent, andimmunoprotective coating around the microcapsules. In this embodiment,the polycation layer is optional because the PEG macrocapsule canreadily provide immunoprotectivity by selection of appropriate molecularweights and concentrations of crosslinkable PEGs.

In yet another presently preferred embodiment, multiplealginate-PLL-alginate microcapsules are entrapped in a macrocapsulecomprising ionically and/or covalently crosslinkable alginate, andcovalently crosslinkable PEG. In this embodiment as well, the polycationlayer is optional because the PEG macrocapsule can readily provideimmunoprotectivity by selection of appropriate molecular weights andconcentrations of crosslinkable PEGs.

In a still further presently preferred embodiment, multiplealginate-PLL-alginate microcapsules are entrapped in an (ionicallyand/or covalently crosslinkable) alginate macrocapsule, followed by acovering of PEG such that PEG and alginate are in intimate contact, andthe PEG is exposed on the surface of the macrocapsule. In thisembodiment as well, the polycation layer is optional because the PEGouter coat can readily provide immunoprotectivity by selection ofappropriate molecular weights and concentrations of crosslinkable PEGs.

In still another presently preferred embodiment, biologically activematerial is entrapped directly in a macrocapsule comprising ionicallyand/or covalently crosslinkable alginate, and/or covalentlycrosslinkable linear or branched chain PEG.

The invention will now be described in greater detail with reference tothe following non-limiting examples.

EXAMPLE 1 Preparation Of PEG Diacrylate Using Acryloyl Chloride

PEG of molecular weight 18500 (abbreviated 18.5 k, also availablecommercially as PEG 20M) was modified chemically sing the followingprocedure to incorporate acrylate functionalities into the moleculewhich rendered it polymerizable. Other PEGs ranging in molecular weightsfrom as low as 200 to as high as (but not limited to) 35000 could alsobe modified by the same procedure.

PEG 18.5 k was dried thoroughly by heating in a vacuum oven at 80° C.for 24 hours. Alternately, the PEG could be dissolved in toluene and thesolution distilled wherein any moisture could be removed as an azeotropewith toluene. 20 g of dry PEG were dissolved in 200-250 ml of drytoluene (acetone, benzene, and other dry organic solvents may also beused). A twofold molar excess of acryloyl chloride was used (0.35 ml)and a base, triethyl amine (0.6 ml) was added to remove HCl uponformation. Before addition of acryloyl chloride, the solution was cooledin an ice bath. The reaction was carried in a round bottomed flask underargon with constant reflux for 24 hours. The reaction mixture wasfiltered to remove the insoluble triethyl amine hydrochloride while thefiltrate was added to an excess of ether to precipitate PE diacrylate.The product was redissolved and precipitated twice for furtherpurification and any remaining solvent removed in vacuum. Otherpurification schemes such as dialysis of a PEG-water solution againstdeionized water followed by freeze drying are also acceptable. Yield: 17g.

EXAMPLE 2 Alternate Methods for Preparation of Polymerizable PEG

Several other methods may be utilized to prepare a modified PEG that ispolymerizable or crosslinkable by introducing unsaturation at the endsof the PEG chain. Some of these methods are briefly outlined below.

An esterification reaction between PEG and acrylic acid (or higherhomologue or derivative thereof) may be carried out in an organicsolvent such as toluene. A small amount of acid such as p-toluenesulfonic acid may be used to catalyze the reaction. Excess of one of thereactants (acrylic acid) will drive the reaction towards the products.The reaction is refluxed for several hours. Since water is formed as aproduct of the reaction, in order to drive the equilibrium towards theproducts, it may be continuously withdrawn by distillation of theazeotrope formed with toluene. Standard purification schemes may beutilized. Methacrylic acid or methacrylate may be reacted in a similarfashion with PEG to obtain the polymerizable derivative (Fukui andTanaka, 1976).

Alternatively, the method of Mathias et al. (1982) which involves thereaction of the PEG alkoxide with acetylene gas to produce the vinylethers of PEG may be employed to produce a polymerizable PEG derivative.

A reaction between PEG and allyl chloride in a dry solvent catalyzed bysmall amounts of stannic chloride also results in a polymerizable PEG.

Several other techniques may be utilized to obtain polymerizable PEGderivatives. Harris (1985) describes a number of protocols involving PEGchemistry from which alternative synthetic schemes are provided.

EXAMPLE 3 Visible Light Photopolymerization To Produce PEG Gels

PEG derivatives prepared by the techniques outlined in examples 1 and 2were dissolved in aqueous bicarbonate buffered saline (such as 5-40 wt%, or other buffer) at pH 7.4. The photosensitizer, ethyl eosin (0.01 μMup to 0.1M), a cocatalyst, triethanolamine (0.01 μM up to 0.1M), andcomonomer, vinyl pyrrolidinone (0.001 to 10%, but not essential) wereadded to the solution which was protected from light until thephotopolymerization reaction. In the alternative, other initiatorscocatalysts, comonomers and wavelengths of laser radiation may be used,the selection of which is well known in the art.

A small quantity of the prepared solution was taken in a test tube andexposed to visible radiation either from an argon ion laser at awavelength of 514 nm at powers between 10 mW to 2 W, or a 100 wattmercury arc lamp which has a fairly strong emission around 514 nm. Thegelling time was noted and found to be extremely rapid with the laser(on the order of milliseconds) and fairly rapid with the mercury lamp(on the order of seconds) and varied with the concentrations of polymer,initiator, cocatalyst, and comonomers in the system.

EXAMPLE 4 UV Light Photopolymerization To Produce PEG Gels

A different initiating system from the one above was used to produce PEGgels. A UV photoinitiator, 2,2-dimethoxy-2-phenyl acetophenone was addedto a solution of polymerizable PEG in aqueous buffer at a concentrationof 1000-1500 ppm. This solution was exposed to long wave UV radiationfrom a 100 watt UV lamp. The time required for gellation was of theorder of seconds and was a function of the concentrations of initiatorand addition of other polymerizable monomers such as vinyl pyrrolidone(0.001 to 10%). A UV laser may also be used for the photopolymerization.Other UV photoinitiators may also be used (e.g., benzoin ethyl ether).

EXAMPLE 5 Geometries of Microcapsule-Containing PEG Gels forImplantation

Microcapsules of alginate or any other material containing cells orenzymes or drugs may be delivered and retrieved from an implant usingvarying geometries of the microcapsule-containing PEG gels. A largenumber of individual microcapsules may be localized to a preferredregion in the peritoneal cavity (or other implantation site) byembedding these microcapsules in a crosslinked PEG gel. Variousgeometries may be considered for implantation such as cylindrical rod,circular disk, flat plate, dog bone, and long cylindrical "laces" or"threads" or "worms."

Regardless of the geometry, the formation of the final implantableproducts required similar processing techniques. The cell containingmicrocapsules were suspended in a physiological solution of PEG 18.5Kdiacrylate (30 wt %; PEGs of varying molecular weights may be used)containing triethanolamine (0.01 μM up to 0.1M), ethyl eosin (0.01 μM upto 0.1M), and vinyl pyrrolidinone (0.001 to 10%). This suspension wasexposed to visible light from a laser or mercury lamp which caused rapidcrosslinking of PEG-diacrylate and resulted in microcapsules embedded inthe PEG gel.

In order to produce a cylindrical "lace," a suspension is disposed in ahypodermic needle having a suitable gauge needle or cannula and theemergent stream is expelled into a buffered solution (which may havemultivalent cations if alginate is used in the suspension) andsimultaneously exposed to laser radiation from a suitable lamp source ifphotopolymerization is required for the reaction. The lamp could be alaser light source or a UV light source depending upon the material tobe polymerized and the photoinitiator used, as is known in the art.Crosslinking is instantaneous, i.e., the emerging stream isphotocrosslinked as fast as it is extruded, resulting in the formationof a "lace" or "noodle." Gels or other geometries can be produced fromappropriate molds fabricated for the purpose.

EXAMPLE 6 Alginate Gel Entrapment Of Microencapsulated Islets

Canine islets were isolated from donor pancreata by collagenasedigestion and purified using a physiological islet purificationsolution. Islets were then encapsulated in an alginate-polylysinealginate microcapsule by the following process: Ten thousand purifiedislets were suspended in 1.8% solution of Na alginate (G content 64%)and via an air-droplet generating device, islets were entrapped inalginate gel beads by crosslinking alginate in 0.8% CaCl₂ solution. Apolylysine membrane was formed following suspension of these alginateencapsulated islets in a 0.1% polylysine solution for 4-8 minutes. Theencapsulated islet was then coated with an outer layer of alginate bysuspension in 0.2% alginate for 5 minutes.

A cylindrical tube (i.e., a lace, approximately 1-5 mm in diameter) ofalginate encasing these microencapsulated islets was then produced asfollows:

The microencapsulated islets were suspended in 1.8% alginate in theratio of 1 volume microcapsule pellet to 4 volumes of alginate, andextruded into a solution of divalent cations containing CaCl₂ alone, ora combination of barium and calcium chloride. The ratio of barium andcalcium chloride as a combination of divalent cations crosslinking agentcould vary from 1:10 to 1:100 (barium:calcium), and the preferred ratiois in the range of 1:50 to 1:100. By combining barium and calcium, a gelof greater strength results. Thus, by this process of ioniccrosslinking, approximately 8000 microencapsulated islets are entrappedin a 16 cm long cylindrical tube of alginate gel.

Alternatively, spherical macrocapsules containing microencapsulatedislets can be prepared by introducing droplets of the above-describedsolution (i.e., microencapsulated islets in alginate solution) into abath containing polyvalent cations (e.g., Ca⁺⁺), as described above.

EXAMPLE 7 Variations in Composition of Retrievable Systems

A number of variations in compositions and materials used in the designof retrievable systems are acceptable and advantageous. The crosslinkedalginate or PEG spheres (or lace) containing alginate microcapsules maybe replaced in the appropriate situations by an ionically and/orcovalently crosslinked alginate spheres. Combinations of alginate andPEG may also be used. The following variations, among others, arepossible:

(i) PEG alone, crosslinked as described above; mixtures of differentmolecular weight crosslinkable PEGs (linear or branched chain) may beused to adjust permeability of the resultant gels;

(ii) Alginate alone, ionically crosslinked;

(iii)Alginate alone, modified to be both ionically and covalentlycrosslinkable;

(iv) A combination of ionically crosslinkable alginate and covalentlycrosslinkable PEG (linear or branched chain);

(v) A combination of modified alginate (i.e., ionically and covalentlycrosslinkable) and covalently crosslinkable PEG (linear or branchedchain); and

(vi) An ionically and/or covalently crosslinked alginate macrocapsule(e.g., a sphere or lace) covered with a coating of covalentlycrosslinked PEG (linear or branched chain) covering the exterior suchthat the PEG and alginate are in intimate contact.

Ionic crosslinking of alginate can be accomplished with either Ca⁺⁺alone or a combination of Ba⁺⁺ and Ca⁺⁺ in a ratio of 1:10 to 1:100(Ba:Ca).

The alginate referred to in the preceding paragraph may be replaced witha modified alginate that is chemically crosslinkable in addition to itsionic gelling capabilities. The modified alginates mentioned herein aredescribed in detail in copending U.S. patent application Ser. No.07/784,267, incorporated by reference herein in its entirety.

The PLL referred to above may also be replaced with a modified PLL thatis covalently crosslinkable. The modified PLL mentioned herein isdescribed in detail in copending U.S. patent application Ser. No.07/784,267, incorporated by reference herein in its entirety.

EXAMPLE 8 Kinetics of Insulin Diffusion from Gel EntrappedMicroencapsulated Islets: In Vitro Studies

Kinetics of insulin secretion from the gel entrapped encapsulated canineislets were compared to individual microencapsulated islets orunencapsulated canine islets as follows: either free unencapsulatedcanine islets (controls) or encapsulated canine islets or gel entrappedencapsulated canine islets were incubated in RPMI culture mediumcontaining a basal level of 60 mg % glucose for 60 minutes, thentransferred to medium containing a stimulatory level of 450 mg % glucosefor 60 minutes and returned to basal medium (60 mg % glucose) for afurther 60 minutes. These tests were performed in triplicate. Thesupernatant was collected at the end of each 60 minute period. Insulinsecretion was assayed by measuring insulin concentration (μU/ml perislet equivalent count) in the supernatant, using RIA.

The study was repeated, but in addition, 10 mM theophylline was added tothe 450 mg % glucose as an added stimulus of insulin secretion. Theresults of these studies are shown in Table 1.

                                      TABLE 1    __________________________________________________________________________    In Vitro Insulin Secretion in Response to Static Glucose Stimulation                    INSULIN (μ/ml/islet/hr)                                INSULIN RATIO                    ACTUAL VALUE                                ABOVE BASAL                    Basal                       Stimulated                            Basal                                Basal                                   Stimulated                                        Basal    __________________________________________________________________________    I.      Unencapsulated Islets    A.  Glucose Stimulated        Mean        3.50                        5.40                            6.10                                1  1.45 1.50        Standard Deviation                    2.55                        4.81                            6.22                                0  0.35 0.71    B.  Glucose + Theophylline        Mean        5.40                       15.45                            11.20                                1  3.00 2.15        Standard Deviation                    1.56                        0.50                            2.26                                0  0.71 0.21    II.      Microencapsulated Islets    A.  Glucose Stimulated        Mean        4.77                        7.03                            5.08                                1  1.50 1.15        Standard Deviation                    3.30                        5.77                            2.80                                0  0.66 0.30    B.  Glucose + Theophylline        Mean        4.01                       21.63                            6.45                                1  5.02 1.35        Standard Deviation                    2.62                       26.36                            4.26                                0  2.86 0.32    III.      Entrapped Microencapsulated      Islets    A.  Glucose Stimulated        Mean        6.85                       17.30                            13.43                                1  2.40 2.00        Standard Deviation                    4.61                       14.43                            9.02                                0  0.80 0.18    B.  Glucose + Theophylline        Mean        5.10                       38.30                            31.68                                1  8.28 6.45        Standard Deviation                    3.41                       26.40                            18.43                                0  6.50 2.22    __________________________________________________________________________

One skilled in the art would expect that entrapping encapsulated canineislets in a further outer coat of polymer material would result indecreased response to a stimulus and reduced diffusion of insulin. Thatis, entrapping encapsulated islets in yet a second layer of polymermaterial the microcapsule plus the macrocapsule! would be expected toreduce the responsiveness of the encapsulated islet to an outsidestimulus. Unexpectedly, according to the present invention, it has beenfound that the rapidity and intensity of the in vitro response of gelentrapped, microencapsulated islets was equal to, if not better than,the free floating encapsulated islets (as shown in Table 1).

Following stimulation with 450 mg % glucose alone, the insulin response(see Table 1) from the microencapsulated islets immobilized in amacrogel ("entrapped MC") was equal to, if not better than the freefloating microencapsulated islets ("Free MC") as well as the freeunencapsulated islet controls ("Free Islets"). This phenomenon was alsoobserved in response to 450 mg % glucose+10 mg theophylline stimulus.While not wishing to be bound to any particular theory, it is currentlyhypothesized that this surprising result is explained by the fact thatinsulin is a negatively charged protein, and with the release of insulinfrom the entrapped microencapsulated cells in response to glucosestimulus, the negatively charged insulin is electrostatically dischargedfrom the negatively charged entrapping gel material, thus acceleratingthe response time of insulin release. Enhancement of insulin releasefrom microencapsulated islets within a macrogel, as compared to insulinrelease from individual encapsulated islets, can also be explained bythe fact that the microcapsule is more neutrally charged than themacrocapsule, due to the presence of the positively charged PLL in themicrocapsule membrane. This unexpected result was corroborated in the invivo studies (described in Example 9) where the fall in serum glucose inresponse to an intravenous glucose injection occurred rapidly indiabetic rats treated with gel entrapped microencapsulated canine islets(see FIG. 3), suggestive that gel entrapment provides an added advantageof improved insulin response to a systemic glucose signal.

The results shown in Table 1 demonstrate that gel entrapment ofencapsulated canine islets did not impair insulin secretion in responseto a glucose stimulus or in response to glucose plus theophylline.Return of insulin levels to basal state was however delayed in themacrocapsule experiment, and is consistent with entrapment of insulin inthe outer gel, resulting in effect a sustained release drug deliverysystem. In in vivo applications, this sustained release of insulin mayprovide a more stable homeostatic mechanism.

Thus gel entrapment of microencapsulated islets provides severalunexpected advantages with regard to insulin secretion:

(i) rapid response of insulin release to a glucose stimulus, and

(ii) once the insulin is released, a slow return to basal state,providing a sustained, continuous release over a longer period.

Both of these responses are important in improving the homeostaticcontrol of glucose metabolism in the diabetic recipient.

EXAMPLE 9 In Vivo Studies of Gel Entrapped Microencapsulated Islets

The efficacy of gel-entrapped microencapsulated canine islets wascompared to free-floating encapsulated canine islets in the treatment ofstreptozoticin-induced (STZ) diabetic Lewis rats. Eight thousandencapsulated canine islets (either free-floating, or entrapped incylindrical tubes of alginate) were implanted into the peritonealcavities of four STZ-diabetic Lewis rats, and compared with eightthousand unencapsulated canine islets (controls) implanted in the sameway.

To ensure no variability in the viability, purity and function of theislet preparation, islets from the same canine pancreatic donor wereused in both free floating microencapsulated and gel entrappedmicroencapsulated groups. Furthermore to ensure no variability in theintegrity and formation of the microcapsules themselves, the islets wereall encapsulated in one batch process and then divided into two--onehalf being used for free floating implantation and the remaining halfentrapped in an alginate macrocapsule (i.e., gel entrappedmicroencapsulated islets) prior to implantation.

Immunoprotectivity, biocompatibility, function and graft survival of theunencapsulated (free) islets compared to free-floating encapsulatedislets (in microcapsules only) compared to entrapped encapsulated islets(in macrocapsules with microcapsules therein) were compared bymeasuring:

(i) Daily serum glucose levels (see FIG. 2)

Diabetes is induced in rats by STZ injection. Injected rats areconsidered diabetic if serum glucose levels are greater than 200 mg %.Unencapsulated canine islets (free islets) failed to restorenormoglycemia (serum glucose levels less than 200 mg %) in the diabeticrat. In contrast, the free floating microencapsulated islets and themacroencapsulated islets both restored normoglycemia in these diabeticrats by the 3rd-4th day. By day 7, the free floating microencapsulatedislets began to fail, with serum glucose levels rising in theserecipients to >200 mg %. In contrast, rats receiving gel entrappedmicrocapsules (macrocapsules) continued their normoglycemic state, withserum glucose levels below 100 mg % (FIG. 2). These in vivo studiestherefore provide evidence that (compared to free floatingmicroencapsulated islets) gel entrapment of microencapsulated isletsprolongs graft survival, presumably by enhancing cytoprotectivity andbiocompatibility.

(ii) daily urine volume

With onset of diabetes, urine volume increases. Following STZ injection,urine volume exceeds 30 ccs per day and is indicative of the diabeticstate. With resolution of diabetes, urine volume falls to normal levels.Reduction in urine volume coincided with the fall in serum glucose,providing further evidence that graft function and survival ofmacroencapsulated islets was superior to both free unencapsulated isletsand free floating microencapsulated islets. On day 7, urine volume infree floating microencapsulated group rose to diabetic levels (>30mls.), indicating onset of graft failure, while the rats receiving gelentrapped microcapsules (i.e., macrocapsules) maintained a normal levelof urine output, indicating ongoing graft function.

(iii) body weight

Following STZ injections, diabetic rats lose weight dramatically.Maintenance of weight or weight gain is an excellent parameter offunction of the transplanted islets. Similar to the correlation betweenurine volume and serum glucose levels, changes in body weightcorroborated the above analysis, providing further evidence that ratsreceiving macroencapsulated islets retained normal graft functioncompared to graft failure in rats receiving free unencapsulated isletsand those receiving free floating microencapsulated islets. Body weightimproved significantly in the animals receiving gel entrappedmicrocapsules (i.e., macrocapsules), while weight loss as a result ofdiabetes continued in the animals with free unencapsulated islets andfell after day 7 in the animals with free floating encapsulated islets.

(iv) glucose response to systemic glucose challenge (intravenous glucosetolerance test, IVGTT; see FIG. 3)

An IVGTT provides an in vivo assessment of the kinetics of insulinrelease in response to an intravenous glucose challenge. By calculatingthe rate of fall of glucose over time, a numerical value (K-value) canbe ascertained and compared to normal non-diabetic rats. The more rapidthe fall in serum glucose following the glucose challenge, the higherthe K-value. The fall in serum glucose in response to an intravenousglucose challenge was studied in both the rats receivingmacroencapsulated and free floating microencapsulated islets. Serumglucose was measured at 1, 4, 10, 20, 30, 60 & 90 minutes following anintravenous injection of 0.5 cc per kg of 50% dextrose. As can be seenin FIG. 3, following the intravenous glucose injection, serum glucoserose to 489 mg % in the macroencapsulated group, and then fell rapidlyto normal levels within 10 minutes, indicating an excellent in vivoinsulin response to the systemic glucose challenge. K-value (whichrepresents the % fall of glucose over time) in these animals was normal(3.81±0.56). This unexpected rapid insulin response corroborates the invitro findings described in Example 8, i.e., that alginate gel entrappedmicrocapsules respond rapidly to a glucose stimulus, and providesfurther evidence that a potential advantage of alginate gel entrapmentis electrostatic repulsion of negatively charged insulin from within thecapsule, and hence rapid physiological fall in glucose in response to asystemic challenge.

The K values of the rats receiving macrocapsules were significantlybetter (K=3.8±0.56) than the rats receiving free floating encapsulatedislets (K=0.6±0.73), indicating loss of graft function in the ratsreceiving free-floating encapsulated islets. Since the isletstransplanted into both groups were from the same canine donor, and thevolume of islets employed were identical, this difference in responsereflects the improved immunoprotectivity of the macrocapsule.

In a separate experiment, free floating encapsulated canine islets weretransplanted into diabetic rats and IVGTT performed in these recipientswhile graft function was normal (serum glucose less than 100 mg %). Ascan be seen in FIG. 4, the serum glucose fell to normal levels followingthe intravenous injection of dextrose only after 30 minutes (K value2.4±0.9) compared to the more rapid fall of glucose (within 10 minutes)in the rats receiving macroencapsulated islets (K value 3.81±0.56).

From the above-described in viva studies, it can be concluded that:

(i) gel entrapment of microencapsulated islets does not decrease therate of diffusion of insulin in response to a glucose stimulus both invitro and in vivo;

(ii) gel entrapment of microencapsulated islets in fact enhances insulinrelease; and

(iii)gel entrapment of microencapsulated islets prolongs graft function.

EXAMPLE 10 Retrieval of Gel Entrapped Microencapsulated Islets

Gel entrapped microencapsulated islets were implanted into STZ induceddiabetic rats and followed for 14 days. Normal serum glucose levels wereachieved within 3 days, and was maintained for the entire observationperiod. On the 14th day, the macrogelled encapsulated islets wereretrieved and examined microscopically.

As observed by photomicrograph, viable intact islets within entrappedmicrocapsules were found with no evidence of cellular overgrowth of theouter surface layer of the macrogel. A photomicrograph (100×) of amacrocapsule containing canine islets according to the present inventionretrieved from the peritoneal cavity of an STZ-diabetic Lewis rat 14days after implantation demonstrates a smooth surface on the outerexposed layer of the macrocapsule, with no evidence of cellularovergrowth. Encapsulated viable individual canine islets can be seencontained within the macrocapsule. At the time of retrieval, thisdiabetic rat was normoglycemic.

Serum glucose levels were monitored in the rat following retrieval ofthe macrogelled encapsulated islets. As can be seen in FIG. 5, within 24hours after retrieval of the macrocapsule serum glucose levels rose togreater than 200 mg %, indicating that the macrogelled encapsulatedislets was responsible for normalization of the serum glucose, andproviding further evidence of the immunoprotectivity andbiocompatibility of the macrocapsule.

From these studies, it can be concluded that:

(i) gel entrapment provides a smoother outer surface, a reduced exposedsurface area and hence increased biocompatibility than free floatingmicroencapsulated islets.

(ii) gel entrapment increases the mechanical stability of encapsulatedislets and provides a retaining matrix even if the individual capsuleswere to break.

EXAMPLE 11 Alleviation of Diabetic Symptoms in a Spontaneous DiabeticDog Implanted with Invention Macrocapsules

A spontaneous diabetic dog having the classical symptoms of Type IDiabetes mellitus at the time of diagnosis, i.e., clinical signs such aspolyuria, polydipsia, polyphagia, weight loss, persistent fastinghyperglycemia (i.e., blood glucose >250 mg/dl), persistent glycosuria,and the need for 1-2 daily injections of insulin to prevent rapiddecompensation, was chosen as a recipient for transplantation of amacrocapsule of the invention (containing a plurality ofmicroencapsulated islets).

Islets from a healthy canine donor were isolated employing conventionalmethods (e.g., collagenase digestion). Islets were thenmicroencapsulated as described above (see, for example, Example 6), inconventional alginate-PLL-alginate microcapsules (approximately 600microns in diameter). The microcapsules were then further entrapped(i.e., encapsulated) into macrocapsules (spherical beads about 4000-8000microns) of ionically crosslinked alginate by extrusion of a suspensionof microcapsules in an alginate solution into a bath containing calciumchloride. Approximately 60-80 microcapsules were entrapped within asingle macrocapsule.

Prior to transplantation, the dog had been maintained on a regimen of 12Units of insulin per day. The transplantation was performed undergeneral anesthesia through a 1.5 ventral midline incision, and themacrocapsules aseptically introduced into the peritoneal cavity througha stainless steel funnel. The dog received a transplant dose of about20,000 islets/kg body weight. The incision was then closed by sutures. Aregimen of prednisolone was maintained for 14 days post-transplant as ananti-inflammatory. Fasting blood glucose, plasma C-peptide, urine outputand body weight were measured daily for the first 14 dayspost-transplant, and every 7 days thereafter. Intravenous glucosetolerance tests (IVGTT) were performed 7 days prior to transplantation,14 and 30 days post-transplantation, and every 30 days thereafter.

All measured parameters returned to normal non-diabetic values withinthree days after transplantation. Blood glucose was maintained wellbelow 200 mg/dl, and the dog remained euglycemic for >150 days followingtransplantation, without the need for any exogenous insulin. IVGTTresults were normal and showed rapid glucose clearance followingchallenge with a bolus of glucose.

EXAMPLE 12 Reversal of Hepatic Deficiency by Transplantation ofEncapsulated Hepatocytes in a Gunn Rat Model

Homozygous Gunn rats having a deficiency of the liver enzyme uridinediphosphate glucuronyl transferase (UDPGT) and resultant elevated serumbilirubin levels (a condition commonly known as jaundice) were chosen asa model of hepatic deficiency. Heterozygous Gunn rats which carry theabnormal gene, but are free from jaundice were chosen as the donors ofhepatocytes for cellular transplant.

The donor liver was cannulated via the portal vein with silicone tubing(i.d. 0.025 inch, o.d. 0.047 inch) and perfused with 100 ml of 0.05%collagenase (Sigma, Type IV) in 15 minutes. The hepatocytes were thenharvested into a sterile beaker and washed three times with RPMI medium.Viability of the isolated hepatocytes was tested by acridinerange/propidium iodide staining and found to be greater than 80%.

The hepatocytes were encapsulated into conventionalalginate-PLL-alginate microcapsules as described above. Thesemicrocapsules were further entrapped (or encapsulated) in sphericalmacrocapsules of ionically crosslinked alginate (as described above.Approximately 50-100 microcapsules were entrapped in a singlemacrocapsule. Each recipient received -10⁷ encapsulated hepatocytes. Themacrocapsules were implanted in the peritoneal cavity of the recipients.

Prior to transplantation, recipient bilirubin levels were 5.3±0.3 mg %.By the second week post-transplantation, serum bilirubin levels werereduced to 2.05±1.05 mg %, which was significantly lower thanpre-transplant levels. These reduced levels were maintained for anaverage of 73±4 days. All recipients received cyclosporin A as ananti-inflammatory (10 mg/kg, i.e., at levels lower than required toprevent immune rejection. Thus, the feasibility and efficacy of themacrocapsule of the invention is demonstrated for in vivotransplantation of hepatocytes.

While the invention has been described in detail with reference tocertain preferred embodiments thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

REFERENCES

Abuchowski et al., 1977; J. Biol. Chem. 252:3578.

Collins et al., U.S. Pat. No. 4,938,961.

Decker and Moussa, 1989; Macromolecules 22:4455.

Desai and Hubbell, 1991a; Biomaterials 12:144-153.

Desai and Hubbell, 1992a; Biomaterials 13:505-510.

Desai and Hubbell, 1992b; Biomaterials 13:417-420.

Soon-Shiong et al., 1991; U.S. Ser. No. 07/784,267.

Dupuy et al., 1988; J. Biomed. Mater. Res. 22:1061.

Duval et al., 1991; Carbohydrate Polymers 15:233.

Eaton, D. F., 1986; Advances in Photochemistry 13:427.

Fukui and Tanaka, 1976; FEBS Letters 66:179.

Fukui et al., 1987; Methods in Enzymology 135:230.

Harris, J. M., 1985; JMS-Rev. Macromol. Chem. Phys. C25:325.

Harris et al., 1984; J. Polym. Sci., Polym. Chem. Ed. 22:341.

Hoyle et al., 1989; Macromolecules 22:3866.

Karu, T. I., 1990, Photochemistry and Photobiology 52:1089.

Kumakura and Kaetsu, 1983; J. Appl. Polym. Sci. 28:2167.

Lim and Sun, 1980; Science 210:908.

Mathias et al., 1982; J. Polym. Sci., Polym. Lett. Ed. 20:473.

Nagoaka and Nakao, 1990; Biomaterials 11:119.

Otterlei et al., 1991; J. Immunotherapy 10:286-291.

Pitha et al., 1979; Eur. J. Biochem. 94:11.

Soon-Shiong et al., 1991; Trans. 3rd Intl. Cong. on Pancreatic & islettransplantation, June 6-8, Lyon, France, p.6.

Soon-Shiong et al., 1991; Transplantation Proc. 23:758.

Weiner et al., 1974, Israel J. Chem. 12:863.

Wu, D. S., November 1990; Laser Focus World, p 99.

Zalipsky et al., 1983, Eur. Polym. J. 19:1177.

Zheng et al., 1991; Transplantation 51:63.

We claim:
 1. A method of treating a patient for diabetes comprisingimplanting a macrocapsule in a defined area of said patient, whereinsaid macrocapsule is formed of a first biocompatible gel and contains aplurality of microcapsules, wherein said microcapsules are formed of asecond biocompatible gel; wherein said microcapsules contain isletcells; and wherein said microcapsules comprise an immunoprotectivecoating.
 2. A method according to claim 1, further comprising removingsaid macrocapsule from said patient when it is no longer effective andreplacing it with a new macrocapsule.
 3. A method according to claim 1,wherein said first and second biocompatible gels are independentlyselected from:a) covalently crosslinkable alginate; b) ionicallycrosslinkable alginate; c) covalently and ionically crosslinkablealginate; d) covalently crosslinkable linear PEG; e) covalentlycrosslinkable branched chain PEG; f) mixtures of different molecularweight covalently crosslinkable linear or branched chain PEG; g)combinations of alginate and PEG; and h) mixtures of any two or morethereof.
 4. A method according to claim 1 wherein said immunoprotectivecoating is selected from the group consisting of polycations, polymerscontaining primary amine groups, polymers containing secondary aminegroups, polymers containing tertiary amine groups, and polymerscontaining pyridinyl nitrogen(s).
 5. A method according to claim 1,wherein said immunoprotective coating is a polycation.
 6. A methodaccording to claim 5, wherein said polycation is a polylysine.
 7. Amethod according to claim 1, wherein said microcapsule has a solid gelcore.
 8. A method according to claim 1, wherein said macrocapsule has asolid gel core.
 9. A method according to claim 1, wherein saidmacrocapsule is formed in the shape of a cylinder, sphere, disc, flatsheet, wafer, or dog-bone.
 10. A method according to claim 1, whereinsaid first and second biocompatible gels are high guluronic acid contentalginate.
 11. A method according to claim 1, wherein said microcapsulesare alginate/PLL/alginate microcapsules.
 12. A method according to claim1, wherein said first and second biocompatible gels are linear orbranched chain polyethylene glycol having a molecular weight rangingfrom 200 to 1,000,000.
 13. The macrocapsule according to claim 1 whereinsaid first biocompatible gel and said second biocompatible gel areindependently selected from the group consisting of PEG, vinyl ethers ofPEG, allyl ethers of PEG, acrylate esters of PEG, and methacrylateesters of PEG.
 14. The macrocapsule according to claim 1 wherein saidfirst biocompatible gel and said second biocompatible gel areindependently selected from the group consisting of alginate, vinylethers of alginate, allyl ethers of alginate, acrylate esters ofalginate, and methacrylate esters of alginate.
 15. A method of treatinga patient for diabetes comprising implanting a macrocapsule in a definedarea of said patient, wherein said macrocapsule comprises a firstbiocompatible gel selected from the group consisting of:a) covalentlycrosslinkable alginate; b) ionically crosslinkable alginate; c)covalently and ionically crosslinkable alginate; d) covalentlycrosslinkable linear PEG; e) covalently crosslinkable branched chainPEG; f) mixtures of different molecular weight covalently crosslinkablelinear or branched chain PEG; g) combinations of alginate and PEG; andh) mixtures of any two or more thereof,wherein said macrocapsule furthercomprises: a plurality of microcapsules, i) wherein said microcapsulescontain islet cells therein; ii) wherein said microcapsules are formedof a second biocompatible gel selected from the group consisting of:a)covalently crosslinkable alginate; b) ionically crosslinkable alginate;c) covalently and ionically crosslinkable alginate; d) covalentlycrosslinkable linear PEG; e) covalently crosslinkable branched chainPEG; f) mixtures of different molecular weight covalently crosslinkablelinear or branched chain PEG; g) combinations of alginate and PEG; andh) mixtures of any two or more thereof; iii) wherein said microcapsulesare each individually treated with an immunoprotective coating, whereinsaid immunoprotective coating is a polycation; and iv) wherein saidsecond biocompatible gel may be the same or different from said firstbiocompatible gel.
 16. A method according to claim 15, wherein saidpolycation is polylysine.
 17. A method according to claim 15, whereinsaid first biocompatible gel is high guluronic acid content alginate.18. A method according to claim 15, wherein said second biocompatiblegel is an alginate, said immunoprotective coating is polylysine, andsaid microcapsules further comprise an alginate outer coat on theoutside of said immunoprotective coating.
 19. A method according toclaim 15, wherein said first and second biocompatible gels are linear orbranched chain polyethylene glycol having a molecular weight rangingfrom 200 to 1,000,000.
 20. A method according to claim 15, wherein saidfirst biocompatible gel comprises covalently crosslinkable linear orbranched chain polyethylene glycol.
 21. A method of treating a patientfor diabetes comprising implanting a macrocapsule in a defined area ofsaid patient, wherein said macrocapsule comprises a first biocompatiblegel containing a plurality of microcapsules therein, wherein eachmicrocapsule comprises:a) a second biocompatible gel containing isletcells therein; and b) optionally an immunoprotective coating surroundingsaid second biocompatible gel;wherein said first and said secondbiocompatible gels may be the same or different, and wherein there is noimmunoprotective coating disposed about the exterior of saidmacrocapsule.